CN108931646B - Test strip for detecting safrole and preparation method and application thereof - Google Patents
Test strip for detecting safrole and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a test strip for detecting safrole, a preparation method and application thereof. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with a safrole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, a safrole monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad, and the safrole hapten is obtained by reacting 5- (2-bromo-2-propenyl-1-yl) -1, 3-benzodioxolane and aminopropionic acid. The invention also provides a preparation method of the safrole test strip and a method for detecting safrole in a sample by applying the test strip. The test strip and the detection method provided by the invention have the advantages of simple operation, high sensitivity, high detection speed, low cost and no limitation of detection equipment, and can realize rapid detection and on-site monitoring of safrole in a large batch of samples.
Description
Technical Field
The invention relates to a test strip for detecting safrole, a preparation method and application thereof, in particular to a colloidal gold test strip for detecting safrole, which is particularly suitable for detecting safrole in tobacco flavors and fragrances, tobacco and tobacco products.
Background
Safrole is also called safrole and safrole, is colorless or yellowish liquid, is insoluble in water, is easily soluble in chloroform, ether and other nonpolar organic solvents, has camphor wood smell, is naturally present in plants such as cinnamon, nutmeg, black pepper, perilla and the like, and is a main component of a plurality of edible natural essential oils such as safrole essential oil, anise essential oil and camphor oil. Due to its pleasant odor, safrole has been used as a flavoring and perfuming agent in food, washing products and cosmetics. The safrole compounds mainly include safrole, isosafrole, dihydrosafrole, etc. Safrole is also an important component of illegal production of drug dancing outreach. As the test shows that the safrole has the effect of causing liver tumor to mice, the limited amount of safrole is regulated in various countries. In 2001, the European Commission regulated the total use limit of safrole and isosafrole to 1 mg/kg in food and beverages and 5 mg/kg in alcoholic beverages. In 2005, the european commission listed safrole as a suspected weak carcinogen, and although the daily limits were not specified, it was required to be set as low as possible. In the 'easy-to-prepare chemical management regulation' implemented in 2005 in China, safrole is listed as the first class of easy-to-prepare chemicals. In the production and manufacturing process of the tobacco essence perfume, the safrole is possibly introduced from the natural plant essential oil, and the essence perfume added in the tobacco shreds is present in tobacco products, so that potential harm is caused to the health of consumers. Therefore, the establishment of a rapid detection method of safrole is necessary to carry out quality control on flavors and fragrances and tobacco products.
Currently, safrole is mainly extracted by a direct extraction method, an ultrasonic extraction method, a simultaneous distillation extraction method, a steam distillation method and the like, and is detected by combining a Gas Chromatography (GC), a gas chromatography-mass spectrometry (GC-MS), a gas chromatography-tandem mass spectrometry (GC-MS/MS), a High Performance Liquid Chromatography (HPLC) and an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Although the instrumental analysis method can realize accurate qualitative and quantitative determination, complex and expensive instruments and equipment and professional operators are needed, and the pretreatment of the sample is complicated, time-consuming and high in detection cost, so that the requirements of field monitoring and large-scale sample screening are difficult to meet. Therefore, it is an urgent problem to develop a product and a method which are not limited by the detection equipment and can realize rapid detection of a large number of samples. At present, the patents and literature reports related to the detection of the safrole in tobacco flavors and tobacco are few, and no safrole test strip appears on the market.
Disclosure of Invention
The invention aims to provide a test strip for detecting safrole and a preparation method and application thereof based on the prior art, and the test strip for detecting safrole in tobacco flavors and fragrances and tobacco products is used for detecting safrole by using a colloidal gold immunochromatography method, has the characteristics of high accuracy, simplicity in operation, no need of professional operation, no need of matching with other detection equipment, convenience in carrying and detection, short time, low detection cost and the like, and is suitable for screening of a large number of samples on site.
In order to achieve the purpose of the invention, the invention provides a test strip for detecting safrole, which comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with a safrole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and a safrole monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad; the safrole monoclonal antibody is prepared by taking a safrole hapten-carrier protein conjugate as an immunogen. The safrole hapten-carrier protein conjugate is obtained by coupling safrole hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or human serum albumin, the safrole hapten is obtained by reacting 5- (2-bromo-2-propylene-1-yl) -1, 3-benzodioxolane and aminopropionic acid, and the molecular structural formula is as follows:
the preparation method of the safrole hapten comprises the following steps:
taking 1.0 g of 5- (2-bromo-2-propen-1-yl) -1, 3-benzodioxolane, adding 50 mL of anhydrous dimethyl sulfoxide (DMSO) for dissolving, stirring for clarification, adding 0.48 g of potassium hydroxide, adding 0.20 g of anhydrous sodium iodide, stirring vigorously, adding 0.41 g of aminopropionic acid, and reacting at 100 ℃ for 4 hours. Stopping the reaction, adding 50 mL of water, adding 1mol/L hydrochloric acid to adjust the pH value to 6, adding 80 mL of ethyl acetate, extracting for three times, combining organic phases, evaporating to dryness by rotary evaporation, loading on a silica gel column, and eluting and separating by using ethyl acetate/petroleum ether with the volume ratio of 1:5 to obtain 0.91g of the hapten product safrole propionate.
The goat anti-mouse anti-antibody is obtained by immunizing a goat by taking a murine antibody as an immunogen.
The sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate, and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.
The bottom plate is a PVC bottom plate; the sample absorption pad is suction filter paper; the conjugate release pad is glass wool; the water absorption pad is absorbent paper; the reaction membrane is a nitrocellulose membrane.
Another object of the present invention is to provide a method for preparing the test strip, which comprises the following steps:
1) preparing a conjugate release pad sprayed with a safrole monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a safrole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
Specifically, the steps include:
1) preparing safrole hapten by reacting 5- (2-bromo-2-propen-1-yl) -1, 3-benzodioxolane with aminopropionic acid;
2) coupling the safrole hapten with carrier protein to prepare a safrole hapten-carrier protein conjugate;
3) immunizing a mouse by using the safrole hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a hybridoma cell strain secreting the safrole monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) coating the safrole hapten-carrier protein conjugate and the goat anti-mouse anti-antibody on a detection line (T) and a quality control line (C) of a reaction membrane respectively;
6) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;
7) adding the prepared safrole monoclonal antibody into the prepared colloidal gold to obtain a safrole monoclonal antibody-colloidal gold marker;
8) spraying the safrole monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 1h, taking out, and storing in a dry environment for later use;
9) soaking the sample absorption pad in 0.1mol/L phosphate buffer solution containing 0.5% bovine serum albumin (mass fraction) and pH of 7.2 for 2h, and drying at 37 ℃ for 2 h;
10) a sample absorbing pad, a conjugate releasing pad, a reaction membrane and a water absorbing pad are sequentially adhered on the bottom plate, and the conjugate releasing pad is covered by the sample absorbing pad from the 1/3 area at the starting end. And finally cutting into small strips with the width of 3mm, adding a plastic box, carrying out vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃. Covering 1/3 of the conjugate release pad with the sample absorption pad can prolong the observation time of the detection result, and make the sample absorption pad sufficiently absorb the detection liquid and sufficiently react with the gold-labeled antibody, thereby reducing errors.
Another object of the present invention is to provide a method for detecting safrole in a sample by using the above test strip, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using a test strip;
(3) and analyzing the detection result.
The test strip for detecting safrole of the invention utilizes antibody-antigen highly specific reaction and immunochromatography analysis technology to fix the safrole monoclonal antibody-colloidal gold marker on the conjugate release pad, and the safrole in the sample is combined with the safrole monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form the safrole-antibody-colloidal gold marker. The safrole in the sample and the safrole hapten-carrier protein conjugate on the reaction film detection line compete to bind with the safrole monoclonal antibody-colloidal gold marker, and whether the sample liquid to be detected contains safrole residues or not is judged according to the depth of a red strip of the detection line.
During detection, a sample is treated and then dropped into a test strip card hole, when the concentration of safrole in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker can be combined with a safrole hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, a red strip is respectively formed at a detection line (T) and a quality control line (C), and the color development of the T line is deeper than that of the C line or is consistent with that of the C line; if the concentration of safrole in the sample is equal to or above the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the safrole, and thus no red band will appear or the color will be lighter than that of line C at line T because the competition reaction will not bind to the safrole hapten-carrier protein conjugate.
Negative: when the quality control line (C) shows a red strip, the detection line (T) also shows a red strip, and the color of the T line is close to or deeper than that of the C line, the result is judged to be negative.
Positive: and when the quality control line (C) shows a red strip, and the detection line (T) does not show color or the color of the T line is lighter than that of the C line, the test line is judged to be positive.
And (4) invalidation: when the quality control line (C) does not show a red strip, the test strip is judged to be invalid whether the detection line (T) shows a red strip or not.
At present, no test strip for detecting the tobacco essence and spice and the safrole in tobacco products exists, and the invention fills the blank. The hapten has proper terminal active groups, the length of a modification site and a spacer arm is selected properly, and the molecular structure of safrole can be simulated to the greatest extent. Meanwhile, the test strip has the advantages of low cost, simple operation, short detection time, no limitation of detection equipment, suitability for various units, simple storage and long quality guarantee period. In conclusion, the method for detecting safrole by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
Fig. 1 is a schematic diagram of a cross-sectional structure of a test strip, in which: 1. a sample absorbing pad; 2. a conjugate release pad; 3. a reaction film; 4. a water absorbent pad; 5. detecting lines; 6. a quality control line; 7. a base plate;
FIG. 2 is a diagram showing the test result of the test strip;
FIG. 3 shows the synthesis of safrole hapten (the figure is used as the abstract figure).
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In addition, various changes or modifications of the present invention may be made by those skilled in the art within the scope defined by the appended claims, and these changes or modifications also fall within the scope of the present invention.
Example 1 preparation of test strip for detecting safrole
The preparation method of the test strip mainly comprises the following steps:
1) preparing a conjugate release pad sprayed with a safrole monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a safrole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a PVC base plate to form the test strip.
The following steps are detailed:
1. synthesis and identification of safrole hapten (the synthetic route is shown in figure 3)
Taking 1.0 g of 5- (2-bromo-2-propen-1-yl) -1, 3-benzodioxolane, adding 50 mL of anhydrous DMSO for dissolving, stirring for clarification, adding 0.48 g of potassium hydroxide and 0.20 g of anhydrous sodium iodide, stirring vigorously, adding 0.41 g of aminopropionic acid, and reacting for 4 hours at 100 ℃. Stopping the reaction, adding 50 mL of water, adding 1mol/L hydrochloric acid to adjust the pH value to 6, adding 80 mL of ethyl acetate, extracting for three times, combining organic phases, evaporating to dryness by rotary evaporation, passing through a silica gel column, and eluting and separating by ethyl acetate/petroleum ether (v/v, 1/5) to obtain 0.91g of the hapten product safrole, wherein the yield is 87.37%.
Taking the hapten, performing nuclear magnetic resonance hydrogen spectrum identification,1H-NMR（CDCl3, 300MHz）δ：11.0（1H, -COOH），6.90（1H, ArH, J=6.864），6.68（1H, ArH），6.76（1H, ArH），6.07（2H, s, -CH2-），4.16（2H, s, -CH2-），3.75（1H, dd, =CH2），3.12（2H, t, -CH2-），2.49 （2H, t, -CH2-, 2.0 (1H, s, -NH-). The existence of peaks in the spectrum, wherein the chemical shift delta =11.0 is the resonance absorption peak of carboxyl hydrogen, and the chemical shift delta =3.12 and 2.49 are the resonance absorption peaks of methylene hydrogen on the spacer arm proves that the spacer arm coupling is successful and the safrole hapten structure is correct.
2. Synthesis and identification of safrole coupling antigen
Immunogen preparation-the coupling of safrole hapten and Bovine Serum Albumin (BSA) gives the immunogen.
Taking 9 mg of safrole hapten, adding 0.3 mL of N, N-dimethylformamide for dissolving, clarifying, adding 8.3 mg of carbodiimide (EDC), stirring for 20 min, adding 7 mg of N-succinimide, and continuously stirring for 30 min to obtain hapten activating solution A; taking 50 mg of BSA, adding 4 mL of 0.8% sodium chloride aqueous solution for dissolving to obtain solution B; slowly dripping the solution A into the solution B, stirring at 4 ℃ for 4h, dialyzing and purifying with 0.02 mol/L PB buffer solution for 3 days to obtain safrole-BSA immunogen, subpackaging, and storing at-20 ℃ for later use.
Preparation of coating antigen-coupling safrole hapten and Ovalbumin (OVA) to obtain the coating antigen.
Taking 6 mg of safrole hapten, adding 0.2 mL of dimethyl sulfoxide to dissolve, adding 20 mu L of triethylamine and 18 mu L of isobutyl chloroformate, uniformly mixing, and reacting at 4 ℃ for 30 min to obtain hapten activating solution A; dissolving OVA 50 mg in 4 mL of 0.8% sodium chloride aqueous solution to obtain solution B; slowly dripping the solution A into the solution B, stirring at 4 deg.C for 4h, dialyzing and purifying with 0.02 mol/L PB buffer solution for 3 days to obtain safrole-OVA coating source, subpackaging, and storing at-20 deg.C for use.
3. Preparation of safrole monoclonal antibody
(1) Animal immunization
Injecting the immunogen obtained in the step 1 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.
(2) Cell fusion and cloning
And (3) fusing Balb/c mouse spleen cells generating specific antibodies with myeloma cells SP20, measuring cell supernatant by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain producing the monoclonal antibody.
(3) Cell cryopreservation and recovery
And (3) preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, subpackaging the cell suspension into freezing tubes, and storing the cells in liquid nitrogen for a long time. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
(4) Preparation and purification of monoclonal antibodies
By adopting an in vivo induction method, Balb/c mice (8 weeks old) are injected with sterilized paraffin oil in the abdominal cavity, hybridoma cells are injected in the abdominal cavity after 7 to 14 days, and ascites is collected after 7 to 10 days. Purifying ascites by octanoic acid-saturated ammonium sulfate method, identifying purity by SDS-PAGE electrophoresis, subpackaging small bottles, and storing at-20 deg.C.
(5) Determination of the potency of monoclonal antibodies
The titer of the antibody is measured to be 1 (50000-100000) by an indirect competition ELISA method.
Indirect competitive ELISA method: coating an enzyme label plate with a safrole hapten-OVA conjugate, adding a safrole standard solution, a safrole monoclonal antibody solution and a horse radish peroxidase labeled goat anti-mouse anti-antibody solution, reacting for 30 min at 25 ℃, pouring out liquid in a hole, washing for 3-5 times with a washing solution, and patting dry with absorbent paper; adding a substrate color developing solution, reacting for 15 min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.
(6) Determination of monoclonal antibody specificity
Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to such antigen analogs, often using cross-reactivity as an evaluation criterion. The smaller the cross-reactivity, the higher the specificity of the antibody.
In the experiment, safrole analogs are serially diluted, are respectively subjected to indirect competitive ELISA with monoclonal antibodies, are prepared into standard curves and are analyzed to obtain IC50Then, the cross-reactivity was calculated as follows:
the results of the cross-reactivity of the antibody with safrole analogs are shown in table 1.
TABLE 1 Cross-reactivity test
| Compound (I) | Cross reaction Rate (%) |
| Safrole | 100 |
| Isosafrole | 13.2 |
| Dihydrosafrole | 6.7 |
| Coumarin compound | ＜1 |
| Vanillin | ＜1 |
| Damascone | ＜1 |
As can be seen from Table 1, the safrole monoclonal antibody has no cross reaction with isosafrole, dihydrosafrole, coumarin, vanillin and damascone.
4. Preparation of goat anti-mouse anti-antibody
The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.
5. Preparation of safrole monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Heating 100 mL of 0.01% chloroauric acid aqueous solution to boiling, adding 4 mL of preheated 1% trisodium citrate aqueous solution accurately under stirring, changing the gold chloroauric acid aqueous solution into gray within 2 min, changing the gray chloroauric acid aqueous solution into wine red, continuously stirring and boiling for 8 min, cooling, adding distilled water to reach a constant volume of 100 mL, and storing at 4 ℃. The prepared colloidal gold is clear and transparent by naked eye observation, has no turbidity, has no floating object on the liquid surface, and has wine red color when observed in sunlight.
(2) Preparation of safrole monoclonal antibody-colloidal gold marker
Under magnetic stirring, 0.2 mol/L potassium carbonate solution is used for adjusting the pH value of the colloidal gold to 7.2 (the pH labeling range of different antibodies is 7-8 and can be changed), the safrole monoclonal antibody is added into the colloidal gold solution according to the standard that 20-50 mu g of the antibody is added into each milliliter of the colloidal gold solution, the mixture is stirred and uniformly mixed, the mixture is kept stand for 10 min at room temperature, 10% BSA is added to ensure that the final mass fraction of the mixture in the colloidal gold solution is 1%, and the mixture is kept stand for 10 min. 12000 r/min, 4 ℃ centrifugation for 40 min, abandoning the supernatant, washing the precipitate twice with a redissolving buffer solution, resuspending the precipitate with the redissolving buffer solution with the volume of 1/10 of the initial volume of the colloidal gold, and standing at 4 ℃ for standby.
Redissolving buffer solution: 0.1-0.3% of BSA, 0.05-0.2% of Tween-80 and 0.02 mol/L of phosphate buffer solution with pH of 7.2.
6. Preparation of conjugate Release pad
The conjugate release pad was soaked in 0.5 mol/L phosphate buffer containing 0.5% BSA (mass fraction), pH7.2, soaked for 1h, and baked at 37 deg.C for 3 h. And (3) uniformly spraying the prepared safrole monoclonal antibody-colloidal gold marker on a conjugate release pad by using a Bio dot film spraying instrument, spraying 0.01 mL of the safrole monoclonal antibody-colloidal gold marker on each 1cm of the conjugate release pad, placing the conjugate release pad in an environment at 37 ℃ (the humidity is less than 20%) for 60 min, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (the humidity is less than 20%) for storage for later use.
7. Preparation of the reaction film
Coating the safrole hapten-ovalbumin conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line.
Coating process: diluting the safrole hapten-ovalbumin conjugate to 1 mg/mL by using a phosphate buffer solution, and coating the safrole hapten-ovalbumin conjugate on a detection line (T) on a nitrocellulose membrane by using a Bio dot membrane instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01 mol/L, pH value of 7.4 phosphate buffer and coated on a control line (C) on a nitrocellulose membrane in an amount of 1.0. mu.L/cm using a Bio dot blot membrane apparatus. And (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.
8. Preparation of sample absorbent pad
The sample absorption pad is soaked in 0.1mol/L phosphate buffer solution containing 0.5 percent of bovine serum albumin (mass fraction) and pH7.2 for 2h, and dried for 2h at 37 ℃ for later use.
9. Assembly of test strips
Sequentially adhering a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC bottom plate; the binder release pad is covered by the sample absorption pad from the 1/3 area at the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC soleplate, and the tail end of the water absorption pad is aligned with the tail end of the PVC soleplate; the reaction membrane is provided with a detection line and a quality control line, and the detection line (T) and the quality control line (C) are strip-shaped strips which are vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; the test paper strip is cut into small strips with the width of 3mm by a machine, and the small strips are arranged in a special plastic card and can be stored for 12 months at the temperature of 4-30 ℃.
EXAMPLE 2 detection of safrole in samples
1. Pretreatment of samples
Weighing 1.0 +/-0.05 g of crushed tobacco sample into a polystyrene centrifuge tube; adding 10 mL of 50% methanol aqueous solution, and sufficiently crushing the mixture by using a homogenizer to obtain a sample solution; and transferring 50 mu L of sample liquid and 450 mu L of deionized water, and uniformly mixing the sample liquid and the deionized water to be detected.
2. Detection with test strip
And (3) taking 100 mu L of sample solution to be detected by a liquid transfer machine, vertically dripping the sample solution into the sample adding hole, timing when the liquid flows, reacting for 10 min, and judging the result.
3. Analyzing the results of the detection
Negative (-): the color development of the T line is darker than that of the C line or is consistent with that of the C line, which indicates that the concentration of safrole in the sample is lower than the detection limit, as shown in FIGS. 2a and 2 b.
Positive (+): the color development of the T-line was lighter than that of the C-line or the T-line was not developed, indicating that the safrole concentration in the sample was equal to or higher than the detection limit, as shown in fig. 2C and 2 d.
And (4) invalidation: the absence of line C indicates an incorrect procedure or the test strip has deteriorated and failed, as shown in FIGS. 2e and 2 f. In this case, the instructions should be read carefully again and retested with a new test strip.
Example 3 sample testing example
1. Limit of detection test
Taking a blank tobacco sample, respectively adding safrole in the blank tobacco sample until the final concentration is 0.1, 0.2 and 0.4 mg/kg, taking a test strip for detection, and repeatedly measuring each sample for three times.
When the test strip is used for detecting a tobacco sample, when the adding concentration of the safrole is 0.1 mg/kg, the test strip shows that the color development of a T line is deeper than that of a C line or is consistent with that of the C line, and the test strip is negative; when the adding concentration of the safrole is 0.2 and 0.4 mg/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the T line is not developed and is positive, which indicates that the detection limit of the test strip on the safrole in the tobacco is 0.2 mg/kg.
2. Test for false positive and false negative rates
Taking 20 parts of tobacco positive samples with known safrole content more than 0.2 mg/kg and 20 parts of tobacco negative samples without safrole, detecting by using three batches of test strips, and calculating the negative and positive rates.
The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the coincidence rate of the positive samples is 100 percent, and the false negative rate is 0; when the negative samples are detected, the results are all negative, and the coincidence rate of the negative samples is 100 percent, and the false positive rate is 0. The test strip for detecting safrole can be used for rapidly detecting the safrole in the tobacco.
3. Specificity test
The safrole test paper is used for detecting 500 mu g/L of isosafrole, dihydrosafrole, coumarin, vanillin and damascone. The result shows that the detection of the fluorescence detector is negative. The test paper strip has no cross reaction to 500 mu g/L of isosafrole, dihydrosafrole, coumarin, vanillin and damascenone.
Claims (6)
1. A test strip for detecting safrole comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the kit is characterized in that the reaction membrane is provided with a detection line coated with a safrole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a safrole monoclonal antibody-colloidal gold marker; the safrole monoclonal antibody is prepared by taking a safrole hapten-carrier protein conjugate as an immunogen; the safrole hapten-carrier protein conjugate is obtained by coupling safrole hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or human serum albumin, the safrole hapten is obtained by reacting 5- (2-bromo-2-propylene-1-yl) -1, 3-benzodioxolane and aminopropionic acid, and the molecular structural formula is as follows:
the preparation reaction process of the safrole hapten is as follows:
the specific preparation method of the safrole hapten comprises the following steps: taking 1.0 g of 5- (2-bromo-2-propen-1-yl) -1, 3-benzodioxolane, adding 50 mL of anhydrous dimethyl sulfoxide to dissolve, stirring for clarification, adding 0.48 g of potassium hydroxide and 0.20 g of anhydrous sodium iodide, stirring vigorously, adding 0.41 g of aminopropionic acid, and reacting for 4 hours at 100 ℃; stopping the reaction, adding 50 mL of water, adding 1mol/L hydrochloric acid to adjust the pH value to 6, adding 80 mL of ethyl acetate, extracting for three times, combining organic phases, evaporating to dryness by rotary evaporation, loading on a silica gel column, and eluting and separating by using ethyl acetate/petroleum ether with the volume ratio of 1:5 to obtain 0.91g of the hapten product safrole propionate.
2. The test strip of claim 1, wherein the sample absorbing pad, the conjugate releasing pad, the reaction membrane and the absorbent pad are sequentially adhered to the base plate.
3. The strip of any one of claims 1 or 2, wherein the conjugate release pad 1/3-1/2 is covered under a sample absorbing pad.
4. The test strip of claim 1, wherein the goat anti-mouse anti-antibody is prepared by immunizing a goat with a murine antibody as an immunogen.
5. A method for preparing the test strip of any one of claims 1 to 4, comprising the steps of:
1) preparing a conjugate release pad sprayed with a safrole monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a safrole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
6. A method for detecting safrole in a sample by using the test strip of any one of claims 1-4, the method comprising the steps of:
1) pretreating a sample;
2) detecting a sample by using the test strip;
3) and analyzing the detection result.
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